A new approach to identify the molecular species in the blood stream responsible for insulin-mimetic activity of organic chelates of the vanadyl (V02+) ion will be developed through application of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy. Since bis(acetylacetonato)oxovanadium(IV), bis(maltolato)oxovanadium(IV), and bis(3-methyl- acetylacetonato)oxovanadium(IV) exhibit markedly enhanced insulin- mimetic activity over that of vanadyl sulfate, these compounds will be characterized by EPR and ENDOR spectroscopy to determine whether specific molecular adducts are formed with the major serum transport proteins albumin, transferrin, or transthyretin. Metabolic assays of insulin mimetic activity will be made with use of differentiated 3T3-L1 adipocytes. The level of insulin-mimetic activity of the chelated complexes and their protein adducts will be compared. Experiments will be carried out to determine whether the stoichiometry of V02+- chelates bound to each of the transport proteins can be correlated with the relative concentrations of the V02+-complex and the transport protein that elicit maximum activity in cell assays. Preliminary results already indicate that bis(acetylacetonate)oxovanadium(IV) binds to albumin as a 1:1 adduct and that this ratio of V02+-complex : albumin in cell assays is associated with maximal insulin-mimetic activity. Initial studies will be directed to determine the influence of V02+- chelates on the relative amounts of radioactive glucose incorporated into glycogen and lipid and how this may differ for insulin and other insulin mimetic agents. The three-dimensional structure of V02+-Chelates both free in solution and bound to serum transport proteins will be determined by ENDOR spectroscopy with use of bis(acetylacetonato)oxovanadium(IV) synthetically enriched with carbon-13 or deuterium. The hyperfine (hf) couplings of the vanadium(IV) center with nearby magnetic nuclei will be analyzed to assign the relative positions of the nuclei with respect to the magnetic axes of the V02+ ion. The coordination structure and ligand geometry of the central V02+ ion for each complex will be modeled according to ENDOR distance constraints. We shall assign by ENDOR the types of protein residues that coordinate the metal ion in binding to proteins and determine whether protein residues have displaced chelate atoms or altered their geometry upon adduct formation. Of particular interest is the recent identification in this laboratory of two reversible ionizations that govern in aqueous solutions a pH dependent_distnbution of these V02+-chelates into four spectrally distinguishable species. Since these protonation processes appear to be associated with-hydrogen bonding of solvent molecules to either equatorial or axial oxygens, they may be important in displacement of chelate by protein residues by perturbation of the tautomeric equilibrium of the acac ligand. We shall therefore endeavor to assign the location of the protonatable group by ENDOR spectroscopy with use of perdeuterated V02+-chelate to remove overlapping resonances of nearby organic hydrogens. Since the V02+- chelates and their protein adducts are paramagnetic, these studies may lead to development of a new spectroscopic probe to characterize macromolecular interactions at the subcellular level that oven glucose uptake and metabolism in cells.